4′‐SCF3‐Labeling Constitutes a Sensitive 19F NMR Probe for Characterization of Interactions in the Minor Groove of DNA

Abstract Fluorinated nucleotides are invaluable for 19F NMR studies of nucleic acid structure and function. Here, we synthesized 4′‐SCF3‐thymidine (T4'-SCF3 ) and incorporated it into DNA by means of solid‐phase DNA synthesis. NMR studies showed that the 4′‐SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4′‐SCF3‐DNA constitute an isolated spin system, offering high 19F NMR sensitivity and excellent resolution of the positioning of T4'-SCF3 within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T4'-SCF3 make it a valuable 19F NMR probe for quantitatively distinguishing diverse DNA structures with single‐nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double‐stranded DNA.

Pyridinium dichromate (PDC, 465 mg, 1.24 mmol) and 500 mg 3 Å molecular sieve was dispersed in 5 mL DCM, and compound 9 (400 mg, 0.62 mmol) was added. After stirring for 3 h at rt, the reaction mixture was filtered through a Celite pad and the filtrate was evaporated.
The resulting residue was dissolved in 40 mL toluene, and p-toluenesulfonic acid (80 mg, w/v = 2%) and 1 mL MeOH was added. After stirring for 1.5 h at rt, another portion of NaBH4 (45 mg, 1.2 mmol) was added and stirring continued for 10 min. Acetic acid was added to tune the pH. The residue was concentrated on vacuum and purified on a silica gel column (4% [v/v] MeOH in DCM) to give 10 (97 mg, 0.29 mmol, 46%) and 10′ (9 mg, 0.025 mmol, 4%) as a white foam.

Oligonucleotide (ODN) synthesis and purification
All DNA oligomers were synthesized on an Applied Biosystems Incorporated 394 oligonucleotide synthesizer using standard DNA synthesis cycle. For incorporating T 4'-SCF 3 , the coupling time was extended to 10 min. After synthesis, solid supports were treated with 1 mL 28% (w/w) aqueous ammonium at room temperature for 16-60 h. The supernatant as then separated from the solid supports, and evaporated to dryness. The obtained crude products

Stability of T 4'-SCF 3 modified oligonucleotides
Generally, 5′-FAM-labeled ODN (0.5 μM) was dissolved in DEPC water. A final concentration of 20 mm buffer was added to establish a given pH (pH 5.2 Sodium Acetate; pH 7.0 sodium phosphate; pH 10.0 borax-sodium hydroxide). Total volume is 100 μL. The mixture was incubated 24 h at 37 ⁰C. Aliquots were taken at appropriate intervals, quenched with 10 μL S7 formamide, and analyzed by 20% denaturing polyacrylamide gel. The gel was visualized using a GE Typhoon Gel Imaging Scanner with excitation at 488 nm and emission at 520 nm.

UV melting measurements
UV thermal scans were performed on UV/VIS Spectrometer Lambda 35 with an optical path of 1 cm. A260 nm was recorded as a function of temperature over the range from 20 to 60 ⁰C with the heating rate at 0.5 ⁰C/min. Prior to the measurements, the solution containing 2.0 μM duplex in a buffer of 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA was heated 5 min in 95 ⁰C, then slowly cooled to room temperature. Tm was determined as the fraction of folded duplex at 50%. In brief, the upper and lower baselines of the melting curve were manually chosen and the median was plotted as the algebraic average of the two baselines. The intercept with the absorbance axis corresponds to the Tm. Thermodynamic parameters were determined according to published method [2][3] . Kinetics. The DNA duplex with a single ribonucleotide incorporated were incubated in the reaction buffer mentioned above at 21 ⁰C. After equilibration for 10 min, 0.05 μM RNase H2 were added. Total reaction volume was 100 μL. Aliquots of 10 μL were removed after incubation.
Experiments were repeated three times and data were analyzed by Prism.

NMR studies of oligonucleotides
NMR experiments were performed on Bruker AVANCE NEO or AVANCE III HD 600 MHz NMR spectrometers equipped with 5 mm 1 H-optimized quadruple resonance cryo probe in the temperature range from 5 to 90 ⁰C in 9/1 H2O/D2O or 100% D2O. NMR samples were prepared by dissolving DNA oligonucleotides in 10 mM sodium phosphate buffer (pH 7.0) with the final concentration in the range of 0.2 to 0.5 mM. Excitation sculpting pulse was used for solvent suppression. Proton NMR spectra were referenced to Sodium trimethylsilyl propanesulfonate (DSS). 2D NOESY spectra were recorded at mixing times between 80, 150, 200 and 400 ms, 2D ROESY at mixing time 200 ms, 2D TOCSY at mixing time 60 ms, DQF-COSY spectra were acquired in 100 % D2O. NMR spectra were processed and analyzed using TopSpin and Sparky
1D 19 F NMR and 2D 19 F/ 1 H HOESY spectra were recorded at a frequency of 376.5 MHz on a Bruker Avance 400 MHz NMR spectrometer equipped with a BBO automatic tuning probe using a zgfhigqn.2 pulse sequence or at a frequency of 564.7 MHz on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a 5 mm 1H-optimized quadruple resonance cryo probe. Typical experimental parameters were chosen as follows: spectral width 20 ppm, acquisition time 0.72 s, pre-scan delay 6.5 μs, receiver gain (RG) 101, number of scans 128.
Prior to Fourier transformation all time domain data was processed with an exponential window function using a line broadening factor of 0.3 Hz. 19 F-resonances were referenced relative to external CCl3F or trifluoro toluene.

NOE-distance restrained molecular dynamics calculations
The distances between interproton in duplex were calculated from 150 ms NOESY spectra.
Amber 20 program was used for simulated annealing (SA) calculations that were performed with random velocity. Tolerance of 0.00005 Å was chosen for the SHAKE algorithm for hydrogen atoms. After that, 10 structures with the lowest energy were subjected to energy minimization with a maximum of 100000 steps of steepest descent. DNA 2.0 software was used to determine helical parameters.